Pressurized Pepsin Digestion in Proteomics: AN AUTOMATABLE ALTERNATIVE TO TRYPSIN FOR INTEGRATED TOP-DOWN BOTTOM-UP PROTEOMICS*

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.In-depth characterization and quantitation of protein isoforms, including post-translationally modified proteins, are challenging goals of contemporary proteomics. Traditionally, top-down (1, 2) and bottom-up (3, 4) proteomics have been two distinct analytical paths for liquid-based proteomics analysis. Top-down proteomics is the mass spectrometry (MS)-based characterization of intact proteins, whereas bottom-up proteomics requires a chemical or enzymatic proteolytic digestion of all proteins into peptides prior to MS analysis. Both strategies have their own strengths and challenges and can be thought of as complementary rather than competing analytical techniques.In a top-down proteomics approach, proteins are usually separated by one- or two-dimensional liquid chromatography (LC) and identified using high performance MS (5, 6). This approach is very attractive because it allows the identification of protein isoforms arising from various amino acid modifications, genetic variants (e.g. single nucleotide polymorphisms), mRNA splice variants, and multisite modifications (7) (e.g. specific histone modifications) as well as characterization of proteolytic processing events. However, there are several challenges that have limited the broad application of the approach. Typically, intact proteins are less soluble than their peptide complement, which effectively results in greater losses during various stages of sample handling (i.e. limited sensitivity). Similarly, proteins above ∼40–50 kDa in size are more difficult to ionize, detect, and dissociate in most high throughput MS work flows. Additionally, major challenges associated with MS data interpretation and sensitivity, especially for higher molecular mass proteins (>100 kDa) and highly hydrophobic proteins (e.g. integral membrane proteins), remain largely unsolved, thus limiting the applicability of top-down proteomics on a large scale.Bottom-up proteomics approaches have broad application because peptides are easier to separate and analyze via LC coupled with tandem mass spectrometry (MS/MS), offering a basis for more comprehensive protein identification. As this method relies on protein digestion (which produces multiple peptides for each protein), the sample complexity can become exceedingly large, requiring several dimensions of chromatographic separations (e.g. strong cation exchange and/or high pH reversed phase) prior to the final LC separation (typically reversed phase (RP)¹ C₁₈), which is oftentimes directly coupled with the mass spectrometer (3, 8). In general, the bottom-up analysis rarely achieves 100% sequence coverage of the original proteins, which can result in an incorrect/incomplete assessment of protein isoforms and combinatorial PTMs. Additionally, the digested peptides are not detected with uniform efficiency, which challenges and distorts protein quantification efforts.Because the data obtained from top-down and bottom-up work flows are complementary, several attempts have been made to integrate the two strategies (9, 10). Typically, these efforts have utilized extensive fractionation of the intact protein separation followed by bottom-up analysis of the collected fractions. Results so far have encouraged us to consider on-line digestion methods for integrating top-down and bottom-up proteomics in a higher throughput fashion. Such an on-line digestion approach would not only benefit in terms of higher sample throughput and improved overall sensitivity but would also allow a better correlation between the observed intact protein and its peptide digestion products, greatly aiding data analysis and protein characterization efforts.So far, however, none of the on-line integrated methods have proven robust enough for routine high throughput analyses. One of the reasons for this limited success relates to the choice of the proteolytic enzyme used for the bottom-up segment. Trypsin is by far the most widely used enzyme for proteome analyses because it is affordable (relative to other proteases), it has been well characterized for proteome research, and it offers a nice array of detectable peptides due to a fairly even distribution of lysines and arginines across most proteins. However, protein/peptide RPLC separations (optimal at low pH) are fundamentally incompatible with on-line trypsin digestion (optimal at pH ∼ 8) (11, 12). Therefore, on-line coupling of trypsin digestion and RPLC separations is fraught with technological challenges, and proposed solutions (12) have not proven to be robust enough for integration into demanding high throughput platforms.Our approach to this challenge was to investigate alternative proteases that may be more compatible with automated on-line digestion, peptide separation, and MS detection. Pepsin, which is acid-compatible (i.e. it acts in the stomach to initially aid in the digestion of food) (13), is a particularly promising candidate. This protease has previously been successfully used for the targeted analyses of protein complexes, hydrogen/deuterium exchange experiments (14, 15), and characterization of biopharmaceuticals (16, 17). Generally, pepsin preferentially cleaves the peptide bond located on the N-terminal side of hydrophobic amino acids, such as leucine and phenylalanine, although with less specificity than the preferential cleavage observed for trypsin at arginine and lysine. The compatibility of pepsin with typical LC-MS operation makes it an ideal choice for the development of novel approaches combining protein digestion, protein/peptide separation, and MS-based protein/peptide identification.To develop an automated system capable of simultaneously capturing top-down and bottom-up data, enzyme kinetics of the chosen protease must be extremely fast (because one cannot wait hours as is typical when performing off-line proteolysis). Another requirement is the use of immobilized enzyme or a low enough concentration of the enzyme such that autolysis products do not obscure the detection of substrate peptides. The latter was a concern when using pepsin because prior hydrogen/deuterium exchange experiments used enzyme:substrate ratios up to 1:2 (18, 19). To test whether or not such a large concentration of pepsin was necessary, we performed pepsin digestion at ratios of 1:20. Many alternative energy inputs into the system were considered for speeding up the digestion. For instance, it has been shown that an input of ultrasonic energy could accelerate the reaction rate of a typical trypsin digestion while using small amounts of a protease (20). Because ultrasonic energy results in an increase of temperature and microenvironments of high pressure, it has been hypothesized that the higher temperature was the component responsible for the enhanced enzyme activity (21). López-Ferrer et al. (22, 23), however, have demonstrated that application of higher pressure with incorporation of a Barocycler alone can make trypsin display faster enzyme kinetics. This phenomenon can easily be integrated with an LC separation (which already operates at elevated pressure) to enable an automatable ultrarapid on-line digestion LC-MS proteomics platform. Herein, we refer to this platform as the fast on-line digestion system (FOLDS) (23). Although FOLDS has been described before using trypsin, here the system is characterized with pepsin, and the results obtained are compared with results attainable with trypsin. Like trypsin, pepsin produced efficient protein digestion in just a few minutes when placed under pressure. Because of the natural maximal activity of pepsin at low pH, the FOLDS can be incorporated with a RePlay (Advion Biosciences, Ithaca, NY) system, and this powerful combination is what ultimately makes the integration of top-down and bottom-up proteomics analyses possible. The integrated analysis begins with a chromatographic separation of intact proteins. The separated proteins are then split into two streams. One stream proceeds directly to the mass spectrometer for MS and/or tandem MS analysis. The second stream is split into a long capillary where the chromatographic separation of the proteins is maintained, but their arrival to the mass spectrometer for detection is delayed. This is in essence the concept of RePlay (24, 25). Herein, we have taken the RePlay a step further by implementing our FOLDS technology into the second split delayed stream of proteins. While these delayed proteins travel down the long and narrow capillary, we exposed them to pepsin where, in combination with the pressure, the proteins are quickly and reproducibly digested. These peptide fragments are subsequently subjected to MS and/or tandem MS analysis. The FOLDS RePlay system allows the rapid and robust incorporation of the integrated top-down bottom-up proteomics work flow with the ability to not only identify proteins but also to sequence multisite/combinatorial PTMs because all detected peptides (from the FOLDS analysis) are confined to the original chromatographic peak of the protein they were derived from. The analysis of protein mixtures using this integrated strategy reduces the total amount of samples required to obtain both the top-down and bottom-up data, increases throughput, and improves protein sequence coverage.     

X-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca(2+)-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 ? resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism.     

Trifluoromethyl group as a pharmacophore: effect of replacing a CF3 group on binding and agonist activity of a glucocorticoid receptor ligand

Compound 1, a potent glucocorticoid receptor ligand, contains a quaternary carbon bearing trifluoromethyl and hydroxyl groups. This paper describes the effect of replacing the trifluoromethyl group on binding and agonist activity of the GR ligand 1. The results illustrate that replacing the CF3 group with a cyclohexylmethyl or benzyl group maintains the GR binding potency. These substitutions alter the functional behavior of the GR ligands from agonists to antagonists. Docking studies suggest that the benzyl analog 19 binds in a similar fashion as the GR antagonist, RU486. The central benzyl group of 19 and the C-11 dimethylaniline moiety of RU486 overlay. Binding of compound 19 is believed to force helix 12 to adopt an open conformation and this leads to the antagonist properties of the non-CF3 ligands carrying a large group at the center of the molecule.     

Denervation of the porcine ovaries performed during the early luteal phase influenced morphology and function of the gonad

We studied both morphology and steroidogenic activity of ovaries in gilts after bilateral surgical denervation performed on day 3 of the estrous cycle. Blood samples were collected from day 4 of the first estrous cycle to day 11 of the subsequent cycle. Denervation resulted in a dramatic reduction in the number or in the disappearance of tyrosine hydroxylase/dopamine-beta-hydroxylase- and/or neuropeptide Y-immunoreactive nerve fibres. On day 11 of the second cycle, the number of follicles (3-6 mm in diameter) was lower (p<0.001) in the denervated ovaries, while corpora lutea were not present. Neurectomy also led to a decrease in the concentrations of progesterone, androstendione and 17beta-estradiol in the follicular fluid originated from small (1-3 mm in diameter) as well as medium-sized follicles (3-6 mm in diameter). Similar to follicular fluid, concentration of androstendione in the follicular wall of medium-sized follicles decreased in experimental gilts in comparison to that of control animals. In addition, plasma concentrations of LH and steroid hormones were lower in the control than in the experimental group. Our results show that denervation of ovaries during the early luteal phase of the estrous cycle in gilts resulted in the changes in both morphology and steroidogenic activity. These results confirm the important role of the peripheral nerves in the function of ovaries.     

Virus-like particles of potato leafroll virus as potential carrier system for nucleic acids

Potato leafroll virus is a member of the polerovirus genus. The isometric virion is formed by a coat protein encapsidating single-stranded, positive-sense, mono-partite genomic RNA with covalently attached viral protein at the 5' end. The coat protein of the virus exists in two forms: i) a 23 kDa protein, the product of the coat protein gene, and ii) a 78 kDa protein, the product of the coat protein gene and an additional open reading frame expressed by read-through of the coat protein gene stop codon. The aim of this work was the expression of potato leafroll virus coat protein-based proteins that would be able to assemble into virus-like particles in insect cells. These modified particles were tested for their ability to encapsidate nucleic acids. Two types of N-terminally His-tagged coat protein constructs were used for the expression in insect cells: one, encoding a 23 kDa protein with the C-terminal amino-acid sequence corresponding to the wild type coat protein and the second with additional clathrin binding domain at the C-terminus. The expression of these two proteins by a recombinant baculovirus was characterized by Western immunoblotting with antibodies directed against potato leafroll virus. The protection or putative encapsidation of nucleic acids by these two coat protein derivatives was shown by DNase I and RNase A protection assays.     

Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.     

Structural and serological characterization of the major glycolipid from Rothia mucilaginosa

Structural studies on the major glycolipid isolated from Rothia mucilaginosa were carried out utilising specific chemical degradation, NMR spectroscopy and matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI TOF-MS). The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-sn-Gro (Man A-Man B-Gro), of which Man B was esterified at O-6 by a fatty acid residue. A second fatty acid substituted the secondary methylene position of the glycerol residue, in contrast to the glycolipid previously found in R. dentocariosa and Saccharopolyspora strains, in which the second fatty acid esterified the primary methylene position of glycerol. Results of the ELISA experiment with rabbit specific antibacterial sera indicate that these two major glycolipids are antigenic, and the patterns of serological reactivity are similar but not identical.     

Automatic formation of hypotheses on the relationships between structure of naphthalene analogs and bioluminescence response of bioreporter <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> HK44

Seven hypotheses on relationships between the structure of naphthalene analogs and bioluminescence response of bioreporter Pseudomonas fluorescens were formulated using GUHA (General Unary Hypotheses Automaton) on a training set of 37 compounds. Prediction of bioluminescence response of 12 new naphthalene analogs was successful in 69 % cases and resulted in rejection of single hypothesis. The results demonstrate applicability of GUHA in structure-activity research, especially for qualitative data.     

Wounding increases salt tolerance in tomato plants: evidence on the participation of calmodulin-like activities in cross-tolerance signalling

Cross-tolerance is the phenomenon by which a plant resistance to a stress results in resistance to another form of stress. It has previously been shown that salt stress causes the accumulation of proteinase inhibitors and the activation of other wound-related genes in tomato plants (Solanum lycopersicum). However, very little is known about how different stresses interact with one another, and which are the signalling components that interrelate the responses triggered by different stress types. In the present work, it is shown that mechanical wounding increases salt-stress tolerance in tomato plants through a mechanism that involves the signalling peptide systemin and the synthesis of JA. Data are also provided indicating that calmodulin-like activities are necessary for the downstream signalling events that lead to cross-tolerance between wounding and salt stress. Finally, evidence was gathered supporting the hypothesis that LeCDPK1, a Ca2+ -dependent protein kinase from tomato previously described in our laboratory, could participate in this cross-tolerance mechanism interrelating the signalling responses to wounding and salt stress.     

Intrastriatal infusion of liver growth factor stimulates dopamine terminal sprouting and partially restores motor function in 6-hydroxydopamine-lesioned rats.

Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and may be useful for neuroregenerative therapies. The aim of this work was to investigate the effects of the intrastriatal (IS) infusion of LGF in the 6-hydroxydopamine rat model of Parkinson's disease. Tyrosine hydroxylase-positive innervation was significantly increased in the dopamine-denervated striatum of rats receiving intrastriatal LGF infusions (160 ng/day/rat x 15 days) as compared with a vehicle-infused group. There was no evidence of dopaminergic neurogenesis in the striatum or substantia nigra in any experimental group at the times studied. However, in those animals undergoing IS-LGF infusion for 48 hr, we found a significant increase in both microglial proliferation and in the number of microglial cells that acquired the ameboid morphology. This is characteristic of activated microglia/macrophages that has been reported to play an important role in dopamine terminal sprouting. In summary, our study shows that IS infusion of LGF stimulates the outgrowth of tyrosine hydroxylase-positive terminals in the striatum of 6-hydroxydopamine-treated rats. As apomorphine-induced rotational behavior was also reduced in these animals, we propose LGF as a novel factor that, when delivered to the striatum, may be useful in the treatment of Parkinson's disease.